Current understanding of the molecular mechanisms that underly Histoplasma pathogenesis remains limited. Unlike opportunistic pathogens, the fungal pathogen Histoplasma capsulatum can cause disease even in immunocompetent hosts by parasitizing phagocytes of the host. Only a few virulence factors have been identified and characterized to date. In this proposal, we will use a forward genetics approach to discover the virulence factors that enable Histoplasma to subvert the defenses of the macrophage, Histoplasma's primary host cell. Random mutants of Histoplasma yeasts will be created using insertional mutagenesis. Mutants will be screened for decreased virulence in macrophages using a transgenic macrophage line and a Histoplasma strain that has been engineered with fluorescence to provide high-throughput screening capability. Mutants will be classified according to the stage at which Histoplasma pathogenesis is blocked by analysis of intramacrophage growth kinetics. The virulence genes represented by each attenuated mutant will be identified by mapping of the mutation. The final collection of virulence-defective mutants will be ranked according to the severity of their impairment, the classification of their pathogenesis defects, and the identity of the virulence gene identities. These rankings will be used to prioritize further characterization of the discovered virulence factors in future studies t define their roles in facilitating Histoplasma survival and growth in host macrophages.